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GBM Derived Gangliosides Induce T Cell Apoptosis through Activation of the Caspase Cascade Involving Both the Extrinsic and the Intrinsic Pathway.

Mahata B, Biswas S, Rayman P, Chahlavi A, Ko J, Bhattacharjee A, Li YT, Li Y, Das T, Sa G, Raychaudhuri B, Vogelbaum MA, Tannenbaum C, Finke JH, Biswas K - PLoS ONE (2015)

Bottom Line: GBM-ganglioside induced activation of the effector caspase-3 along with both initiator caspases (-9 and -8) in T cells while both the caspase-8 and -9 inhibitors were equally effective in blocking apoptosis (60% protection) confirming the role of caspases in the apoptotic process.Ganglioside-induced T cell apoptosis did not involve production of TNF-α since anti-human TNFα antibody was unable to protect T cells from nuclear blebbing and subsequent cell death.Thus, GBM-ganglioside can mediate T cell apoptosis by interacting with the TNF receptor followed by activation of both the extrinsic and the intrinsic pathway of caspases.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine, Bose Institute, Kolkata, India.

ABSTRACT
Previously we demonstrated that human glioblastoma cell lines induce apoptosis in peripheral blood T cells through partial involvement of secreted gangliosides. Here we show that GBM-derived gangliosides induce apoptosis through involvement of the TNF receptor and activation of the caspase cascade. Culturing T lymphocytes with GBM cell line derived gangliosides (10-20 μg/ml) demonstrated increased ROS production as early as 18 hrs as indicated by increased uptake of the dye H2DCFDA while western blotting demonstrated mitochondrial damage as evident by cleavage of Bid to t-Bid and by the release of cytochrome-c into the cytosol. Within 48-72 hrs apoptosis was evident by nuclear blebbing, trypan blue positivity and annexinV/7AAD staining. GBM-ganglioside induced activation of the effector caspase-3 along with both initiator caspases (-9 and -8) in T cells while both the caspase-8 and -9 inhibitors were equally effective in blocking apoptosis (60% protection) confirming the role of caspases in the apoptotic process. Ganglioside-induced T cell apoptosis did not involve production of TNF-α since anti-human TNFα antibody was unable to protect T cells from nuclear blebbing and subsequent cell death. However, confocal microscopy demonstrated co-localization of GM2 ganglioside with the TNF receptor and co-immunoprecipitation experiments showed recruitment of death domains FADD and TRADD with the TNF receptor post ganglioside treatment, suggesting direct interaction of gangliosides with the TNF receptor. Further confirmation of the interaction between GM2 and TNFR1 was obtained from confocal microscopy data with wild type and TNFR1 KO (TALEN mediated) Jurkat cells, which clearly demonstrated co-localization of GM2 and TNFR1 in the wild type cells but not in the TNFR1 KO clones. Thus, GBM-ganglioside can mediate T cell apoptosis by interacting with the TNF receptor followed by activation of both the extrinsic and the intrinsic pathway of caspases.

No MeSH data available.


Related in: MedlinePlus

TALEN mediated targeted disruption of TNFR1 gene abolished the ganglioside GM2–TNFR1 interaction in Jurkat-T cells.Fig 7A shows schematic representation of TNFR1 specific TALEN pair. DNA sequence with black letters indicates TALEN target sequence against which TALEN pair has been designed, red letters represent spacer DNA sequences. TALEN modules are represented as yellow, red, green or blue boxes according to their base recognition specificity of A, T, G or C respectively. Large red box with overhanging 3 arrows indicates wild type Fok1 nuclease domain, shown in Fig 7A. Western immunoblotting was performed to check the expression level of TNFR1 transfected, G418 selected Jurkat-T cells vs wild type Jurkat-T cells. β-actin was used as loading control as shown in Fig 7B. Jurkat-T cells treated with GM2 or not for 10hrs were attached in poly-L-lysine coated coverslips and stained with GM2 specific and TNFR1 specific antibodies, counterstained with respective fluorescent tagged secondary antibodies and mounted on slides with Vectashield mounting media containing DAPI and assessed for co-localization of TNFR1 and GM2 under confocal microscope (Fig 7C). Both wild type and TNFR1 KO cells were treated with GM2 of not for 10hrs and processed as described above and visualized under confocal microscope as shown in Fig 7D. Scale bar represents 10μm.
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pone.0134425.g007: TALEN mediated targeted disruption of TNFR1 gene abolished the ganglioside GM2–TNFR1 interaction in Jurkat-T cells.Fig 7A shows schematic representation of TNFR1 specific TALEN pair. DNA sequence with black letters indicates TALEN target sequence against which TALEN pair has been designed, red letters represent spacer DNA sequences. TALEN modules are represented as yellow, red, green or blue boxes according to their base recognition specificity of A, T, G or C respectively. Large red box with overhanging 3 arrows indicates wild type Fok1 nuclease domain, shown in Fig 7A. Western immunoblotting was performed to check the expression level of TNFR1 transfected, G418 selected Jurkat-T cells vs wild type Jurkat-T cells. β-actin was used as loading control as shown in Fig 7B. Jurkat-T cells treated with GM2 or not for 10hrs were attached in poly-L-lysine coated coverslips and stained with GM2 specific and TNFR1 specific antibodies, counterstained with respective fluorescent tagged secondary antibodies and mounted on slides with Vectashield mounting media containing DAPI and assessed for co-localization of TNFR1 and GM2 under confocal microscope (Fig 7C). Both wild type and TNFR1 KO cells were treated with GM2 of not for 10hrs and processed as described above and visualized under confocal microscope as shown in Fig 7D. Scale bar represents 10μm.

Mentions: To investigate whether shed gangliosides interact directly with the TNFRI, co-localization studies were performed on T cells incubated with CCF52 derived gangliosides for 24hrs. Immunofluorescent staining with rabbit anti-human TNFRI antibody (Red) and hamster anti-human GM2 antibody (Green) followed by confocal microscopy showed presence of TNFRI in CCF52 treated T cells as well as the control cells (Fig 6D, panel 1) as evidenced from the red fluorescence. GM2 was also shown to be taken up by T cells treated with CCF52 ganglioside at 24hrs, as observed from the green fluorescence in CCF52 treated T cells versus the control cells (Fig 6D, panel 2). An overlay of green and red channels confirm co-localization of GM2 and TNFRI on the T cells treated with CCF52 ganglioside as observed from the orange or yellow fluorescence, characteristic of co-localization (Fig 6D, panel 3). A normal rabbit IgG antibody was used as the isotype control for TNFRI. This co-localization was further validated and confirmed in Jurkat T cell clones (TNFR1 KO), where the exon 2 of TNFR1 gene locus was mutated by targeted genome editing using TALEN technology. TALEN constructs were obtained which were designed in a way to effectively target the TNFR1 exon 2 as shown in Fig 7A. Transfection of Jurkats with left and right TALEN pairs caused targeted editing of the TNFR1 gene, thereby generating TNFR1 KO clone. The TNFR1 clone shows negligible expression of TNFR1 protein expression as validated by western blot analysis shown in Fig 7B. Both wild type and TNFR1 KO Jurkats were then treated or not with exogenous GM2 at time points much earlier than that required for apoptosis induction, to demonstrate co-localization of GM2 with the TNFR1 receptor as shown in Fig 7C and 7D. Data shows co-localization of GM2 (green) with the TNFR1 (red) in wild type Jurkat cells after 10 hrs of GM2 treatment, as evidenced by the yellow fluorescence in Fig 7C. This was further confirmed in another experiment, where co-localization between GM2 and TNFR1 was observed in wild type Jurkat cells versus TNFR1 KO Jurkats, which showed no co-localization because of negligible TNFR1 expression (Fig 7D).


GBM Derived Gangliosides Induce T Cell Apoptosis through Activation of the Caspase Cascade Involving Both the Extrinsic and the Intrinsic Pathway.

Mahata B, Biswas S, Rayman P, Chahlavi A, Ko J, Bhattacharjee A, Li YT, Li Y, Das T, Sa G, Raychaudhuri B, Vogelbaum MA, Tannenbaum C, Finke JH, Biswas K - PLoS ONE (2015)

TALEN mediated targeted disruption of TNFR1 gene abolished the ganglioside GM2–TNFR1 interaction in Jurkat-T cells.Fig 7A shows schematic representation of TNFR1 specific TALEN pair. DNA sequence with black letters indicates TALEN target sequence against which TALEN pair has been designed, red letters represent spacer DNA sequences. TALEN modules are represented as yellow, red, green or blue boxes according to their base recognition specificity of A, T, G or C respectively. Large red box with overhanging 3 arrows indicates wild type Fok1 nuclease domain, shown in Fig 7A. Western immunoblotting was performed to check the expression level of TNFR1 transfected, G418 selected Jurkat-T cells vs wild type Jurkat-T cells. β-actin was used as loading control as shown in Fig 7B. Jurkat-T cells treated with GM2 or not for 10hrs were attached in poly-L-lysine coated coverslips and stained with GM2 specific and TNFR1 specific antibodies, counterstained with respective fluorescent tagged secondary antibodies and mounted on slides with Vectashield mounting media containing DAPI and assessed for co-localization of TNFR1 and GM2 under confocal microscope (Fig 7C). Both wild type and TNFR1 KO cells were treated with GM2 of not for 10hrs and processed as described above and visualized under confocal microscope as shown in Fig 7D. Scale bar represents 10μm.
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Show All Figures
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pone.0134425.g007: TALEN mediated targeted disruption of TNFR1 gene abolished the ganglioside GM2–TNFR1 interaction in Jurkat-T cells.Fig 7A shows schematic representation of TNFR1 specific TALEN pair. DNA sequence with black letters indicates TALEN target sequence against which TALEN pair has been designed, red letters represent spacer DNA sequences. TALEN modules are represented as yellow, red, green or blue boxes according to their base recognition specificity of A, T, G or C respectively. Large red box with overhanging 3 arrows indicates wild type Fok1 nuclease domain, shown in Fig 7A. Western immunoblotting was performed to check the expression level of TNFR1 transfected, G418 selected Jurkat-T cells vs wild type Jurkat-T cells. β-actin was used as loading control as shown in Fig 7B. Jurkat-T cells treated with GM2 or not for 10hrs were attached in poly-L-lysine coated coverslips and stained with GM2 specific and TNFR1 specific antibodies, counterstained with respective fluorescent tagged secondary antibodies and mounted on slides with Vectashield mounting media containing DAPI and assessed for co-localization of TNFR1 and GM2 under confocal microscope (Fig 7C). Both wild type and TNFR1 KO cells were treated with GM2 of not for 10hrs and processed as described above and visualized under confocal microscope as shown in Fig 7D. Scale bar represents 10μm.
Mentions: To investigate whether shed gangliosides interact directly with the TNFRI, co-localization studies were performed on T cells incubated with CCF52 derived gangliosides for 24hrs. Immunofluorescent staining with rabbit anti-human TNFRI antibody (Red) and hamster anti-human GM2 antibody (Green) followed by confocal microscopy showed presence of TNFRI in CCF52 treated T cells as well as the control cells (Fig 6D, panel 1) as evidenced from the red fluorescence. GM2 was also shown to be taken up by T cells treated with CCF52 ganglioside at 24hrs, as observed from the green fluorescence in CCF52 treated T cells versus the control cells (Fig 6D, panel 2). An overlay of green and red channels confirm co-localization of GM2 and TNFRI on the T cells treated with CCF52 ganglioside as observed from the orange or yellow fluorescence, characteristic of co-localization (Fig 6D, panel 3). A normal rabbit IgG antibody was used as the isotype control for TNFRI. This co-localization was further validated and confirmed in Jurkat T cell clones (TNFR1 KO), where the exon 2 of TNFR1 gene locus was mutated by targeted genome editing using TALEN technology. TALEN constructs were obtained which were designed in a way to effectively target the TNFR1 exon 2 as shown in Fig 7A. Transfection of Jurkats with left and right TALEN pairs caused targeted editing of the TNFR1 gene, thereby generating TNFR1 KO clone. The TNFR1 clone shows negligible expression of TNFR1 protein expression as validated by western blot analysis shown in Fig 7B. Both wild type and TNFR1 KO Jurkats were then treated or not with exogenous GM2 at time points much earlier than that required for apoptosis induction, to demonstrate co-localization of GM2 with the TNFR1 receptor as shown in Fig 7C and 7D. Data shows co-localization of GM2 (green) with the TNFR1 (red) in wild type Jurkat cells after 10 hrs of GM2 treatment, as evidenced by the yellow fluorescence in Fig 7C. This was further confirmed in another experiment, where co-localization between GM2 and TNFR1 was observed in wild type Jurkat cells versus TNFR1 KO Jurkats, which showed no co-localization because of negligible TNFR1 expression (Fig 7D).

Bottom Line: GBM-ganglioside induced activation of the effector caspase-3 along with both initiator caspases (-9 and -8) in T cells while both the caspase-8 and -9 inhibitors were equally effective in blocking apoptosis (60% protection) confirming the role of caspases in the apoptotic process.Ganglioside-induced T cell apoptosis did not involve production of TNF-α since anti-human TNFα antibody was unable to protect T cells from nuclear blebbing and subsequent cell death.Thus, GBM-ganglioside can mediate T cell apoptosis by interacting with the TNF receptor followed by activation of both the extrinsic and the intrinsic pathway of caspases.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine, Bose Institute, Kolkata, India.

ABSTRACT
Previously we demonstrated that human glioblastoma cell lines induce apoptosis in peripheral blood T cells through partial involvement of secreted gangliosides. Here we show that GBM-derived gangliosides induce apoptosis through involvement of the TNF receptor and activation of the caspase cascade. Culturing T lymphocytes with GBM cell line derived gangliosides (10-20 μg/ml) demonstrated increased ROS production as early as 18 hrs as indicated by increased uptake of the dye H2DCFDA while western blotting demonstrated mitochondrial damage as evident by cleavage of Bid to t-Bid and by the release of cytochrome-c into the cytosol. Within 48-72 hrs apoptosis was evident by nuclear blebbing, trypan blue positivity and annexinV/7AAD staining. GBM-ganglioside induced activation of the effector caspase-3 along with both initiator caspases (-9 and -8) in T cells while both the caspase-8 and -9 inhibitors were equally effective in blocking apoptosis (60% protection) confirming the role of caspases in the apoptotic process. Ganglioside-induced T cell apoptosis did not involve production of TNF-α since anti-human TNFα antibody was unable to protect T cells from nuclear blebbing and subsequent cell death. However, confocal microscopy demonstrated co-localization of GM2 ganglioside with the TNF receptor and co-immunoprecipitation experiments showed recruitment of death domains FADD and TRADD with the TNF receptor post ganglioside treatment, suggesting direct interaction of gangliosides with the TNF receptor. Further confirmation of the interaction between GM2 and TNFR1 was obtained from confocal microscopy data with wild type and TNFR1 KO (TALEN mediated) Jurkat cells, which clearly demonstrated co-localization of GM2 and TNFR1 in the wild type cells but not in the TNFR1 KO clones. Thus, GBM-ganglioside can mediate T cell apoptosis by interacting with the TNF receptor followed by activation of both the extrinsic and the intrinsic pathway of caspases.

No MeSH data available.


Related in: MedlinePlus